Aminopeptidase When grown to high density, P. aeruginosa secretes large quantities of a Zn-dependent Leu-aminopeptidase. Although the fully annotated sequences of several Pseudomonas genomes have been reported, this enzyme is found only in P. aeruginosa. Also, Leu-aminopeptidase is known to be up-regulated during biofilm formation. It is therefore under consideration as a novel virulence factor. Small non-coding RNA We have discovered a density dependent small non-coding RNA (of approximately 90 bases) that appears to be associated with the quorum sensing pathway of P. aeruginosa. Currently we are seeking the molecular targets of this RNA. Pseudomonas exotoxin and Immuntoxins Pseudomonas exotoxin (PE) is a soluble highly cytotoxic protein secreted by P. aeruginosa in response to low environmental iron. The toxin is lethal for most mammalian cells because of its ability to enter them by receptor-mediated endocytosis and proceed in retrograde fashion to the ER. From the ER an enzymatically active C-terminal fragment of the toxin translocates to the cell cytosol and ADP-ribosylates elongation factor-2. This is lethal for the cell. We have used the enzymatic and translocation functions of the toxin in combination with antibody fragments to target and kill cancer cells that express specific cell surface antigens. These antibody-toxin proteins are termed "immunotoxins". BL22, one of these immunotoxins is currently being tested in Phase II trials in patients with B-cell malignancies. We study the interaction of PE with eukaryotic cells to learn more about the pathway of this important cell killing protein. Recently, we published on the identification of a second cell surface receptor, LRP1B. We have begun to express enzymatically inactive (non toxic) versions of the toxin in mammalian cells and yeast as a way to dissect the pathway of killing. Specifically, we are attempting to identify key intracellular components that participate in the toxin translocation pathway. Vaccine development To prevent P. aeruginosa infections in Cystic Fibrosis-affected individuals, we have designed a vaccine to inhibit colonization of airway epithelia. The vaccine is composed of a non-toxic form of Pseudomonas exotoxin with an insert derived from a key portion of type IV pili. Typical antibody responses include titers to both the exotoxin (a known soluble virulence factor) and type IV pili (a known bacterial adherence organelle). Final preclinical characterization of the vaccine has been completed and a clinical trial was recently initiated (Spring 2005) sponsored by Trinity Biosytems, Menlo Park CA. Recently, we expanded our vaccine development effort to include key sequences from the coat proteins of human and bovine papilloma virus isolates. Biodefense Botulinum neurotoxin relies on the presence of four poorly characterized accessory proteins to survive stomach acid, to avoid protease digestion in the intestinal tract and to cross the epithelial barrier. To study the role of these four accessory proteins we have cloned and expressed each one individually. Genes for HA17, HA33, HA70 and NTNH were produced synthetically and all four proteins have been successfully expressed in E. coli.